Accurate and rapid diagnosis is one of the important factors in disease management. In particular, rapid diagnosis is very important in the early prevention of infectious diseases such as swine flu, avian influenza, malaria or dengue fever. As an example, many people around the world suffered or died from swine flu or avian influenza infections that occurred in 2009, and this is due to the fact that these infectious pathogens are highly contagious and cause symptoms that can threaten lives (Dawood F S et al., Emergence of a novel swine-origin influenza A (H1N1) virus in human, N Engl J Med, 360(25), pp 2605-2615, 2009; Beigel J H et al., Avian influenza A (H5N1) infection in human, N Engl J Med, 353(13), pp 1374-1385, 2005). In order to prevent the spread of these various infectious diseases in advance, it is required to build a system capable of transmitting the results quickly diagnosed in the field to a central management system in order to systematically monitor the results. In order to rapidly diagnose diseases in the field, it is required to develop biosensors and the like having increased diagnosis accuracy and sensitivity.
Fluorescence technologies have been widely used in various apparatuses for analysis/diagnosis that are utilized in research and clinical diagnoses in the field of biology and medicine (Lian W et al., Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles, Anal Biochem, 334(1), pp 135-144, 2004). This is due to the fact that fluorometry is one of the most sensitive methods that can detect the corresponding organic substances or inorganic substances from an analyte even at a very low concentration. A fluorescent substance-molecule conjugation product (fluorescent bioconjugate) prepared through a simple chemical reaction enables sensitive and quantitative detection of a target substance from an analyte. As a result, numerous organic and inorganic fluorescent substances have been developed to lower costs, secure stability and increase diagnosis sensitivity. However, fluorescent substances that have been developed so far or existing fluorescent substances have a limitation in their use as a biosensor for diagnosis since they still have not overcome deficiencies such as the brightness of a fluorescent substance, the stability of fluorescence duration or severe interference of fluorescence signals. In particular, there is also a disadvantage in that high energy sources are required in order for these fluorescent substances to emit, and a laser diode (LD) always needs to be used.
A laser diode (LD) is a light source that has been used in various analysis apparatuses including diagnostic apparatuses for a long time. However, a laser diode (LD) is more expensive, more inconvenient in operation, has shorter light source life span, and also has a relatively limited light emission range (a wavelength range of approximately 600 nm to 780 nm or 800 nm) compared to a light emitting diode (LED) light source (Imasaka T, Diode lasers in analytical chemistry, Talanta, 48(2), pp 305-320, 1999). In contrast, a light emitting diode (LED) is considered to be a much more efficient light source compared to a laser diode (LD) since an LED has lower costs, stable energy output even with small energy input, longer life span, a very wide light emission wavelength band (a wavelength range of approximately 390 nm to 750 nm, which includes almost the entire wavelength range of an LD). Accordingly, novel fluorescence microscopes for diagnosis that are more economical and have improved diagnosis sensitivity compared to existing microscopes are being developed by replacing the light source of existing fluorescence microscopes for diagnosis with a light emitting diode (LED) (Marais B J et al., Use of light-emitting diode fluorescence microscopy to detect acid-fast bacilli in sputeum, Clin Infect Dis, 47(2), pp 203-207, 2008; Miller A R et al., Portable, battery-operated, low-cost, bright field and fluorescence microscope, Plos One, 5(8), e11890, 2010). Therefore, the development of more efficient, stable and innovative LED-based biosensors for field diagnosis needs to accompany the development of novel fluorescent substances that can emit light using an LED light source with improved signal intensity and stability.
In view of the above, the inventors have developed a novel coumarin derivative multi-fluorescent substance having very improved fluorescence brightness, signal intensity and signal stability.
In addition, the inventors have carried out a fluorescence immunoassay for malaria, which is one of five main diseases highlighted by the World Health Organization (WHO) and a fatal disease with a mortality rate of 5 million worldwide per year, in order to prove the possibility of applying the developed novel coumarin derivative multi-fluorescent substance to the development of an LED-based microfluorescent quantitative biosensor for field diagnosis, that is, to prove its diagnostic usefulness.
A diagnostic method well suited for rapid field diagnosis is an immunochromatographic test (ICT), and most of these have been developed in the form of a dipstick kit. The most widely used dipstick kit for malaria diagnosis worldwide uses a method of detecting plasmodium lactate dehydrogenase (pLDH) and plasmodium histidine rich protein-2 (pHRP-2) antigen among the specific antigens of malaria. As commercialized products utilizing this method, three products including ICT™ Malaria Pf/Pv (Amrad ICT, Australia), OptiMAL (Flow Inc., U.S.A), and ParaSight™ F (Becton Dickinson, U.S.A) are mainly used, and they all have different target antigens. Chansuda investigated these 3 commercialized products, (Wongsrichanalai, C, Rapid diagnostic techniques for malaria control, Trends Parasitol, 17(7), pp 307-309, 2001), and found out that, while these products have high diagnostic sensitivity of approximately 88% to 98% for Plasmodium falciparum, there are problems in that they have diagnostic sensitivity of approximately 75%, 83% and 87%, respectively, for Plasmodium vivax. Different from the assertions of the product suppliers, supplementation is necessary since the diagnostic sensitivity is lower than the sensitivity of existing methods, and in particular, the diagnostic sensitivity for Plasmodium vivax is only 70 to 80% approximately (Cooke A H. et al., Comparison of a parasite lactate dehydrogenase-based immunochromatographic antigen detection assay (OptiMAL) with microscopy for the detection of malaria parasites in human blood samples, Am J Trop Med Hyg, 60(2), pp 173-176, 1999). In addition, Korean Plasmodium vivax often has a long incubation period of normally 6 months to 1 year, as long as 3 years or longer, although it sometimes has a short incubation period of 1 month or less. When this prolonged incubation type occurs, it is difficult to diagnose since it may occur as a subclinical case since the antibody is not formed, or the amount of the antigen is very small. Therefore, there have been demands for the development of sensitive and accurate immunological diagnostic methods capable of diagnosing small amounts of antigens, in addition to the development of novel antibodies capable of early diagnosing a disease.
In view of the above, in order to prepare specific monoclonal antibodies for specific antigens of Plasmodium falciparum (PfMSP, PfLDH, PfHRP2), specific antigens of Plasmodium vivax (PfMSP, PvLDH, PvHRP2), and antigen determinants, the inventors first produced monoclonal antibodies using polypeptide encoding the LDH genes of Plasmodium falciparum, investigated its characteristics, and have verified that diagnosis sensitivity in diagnosing Plasmodium vivax and Plasmodium falciparum patients can be improved through the production of the monoclonal antibodies for specific antigens and antigen determinants (Korean Patent Application Laid-Open Publication No. 10-2006-0027803).
Furthermore, in order to improve the diagnostic methods for the signs of malaria infection, the inventors have used a fluorescence immunoassay (FIA) that has high sensitivity and enables quantitative analysis, and also enables the development of biosensors for field diagnosis on the basis of these performances.
The inventors have prepared a novel compound of coumarin series as a fluorescent substance to be used in a fluorescence immunoassay, and, considering the problem of low fluorescence detection when only one fluorescent binds to an antibody, have invented the novel coumarin derivative multi-fluorescent substance of the present invention, which is designed such that one molecule has a plurality of fluorescent substances by introducing a linker having a core structure, in order to bind a plurality of fluorescent substances to an antibody.
In addition, the inventors have completed the present invention by verifying that a malaria infection can be effectively diagnosed when conjugating the multi-fluorescent substance to a malaria-specific antibody and using a fluorescence immunoassay (FIA).
Furthermore, the inventors have completed the present invention by verifying that a malaria infection can be diagnosed in the field rapidly and in a quantitative way when conjugating the multi-fluorescent substance to a malaria-specific antibody and using a rapid fluorescent immunochromatographic test (FICT).